2.14. Immunofluorescent Assay
Frozen sections were fixed and permeated at room temperature, then incubated with primary antibody against P16 (diluted in goat serum, 1:100) overnight at 4°C. Afterwards, the sections were flushed and incubated with fluorescent secondary antibody diluted with goat serum (1:200) at room temperature for 60 min in the dark. Finally, DAPI was used to stain the cell nucleus for 10 min. The sections were examined under a fluorescence microscope at 200x magnification (DMI4000B; Leica, Wetzlar, Germany).